Models of malignant brain cancer, and therapeutic siRNAs against oncogenic signaling pathways, and methods and kits for uses therefor

Abstract

Methods for screening compounds to treat an oncological disorder regulated through a tumor-inducing pathway are provided. The compounds are administered to non-human animal subjects having a disease model, so that the subjects display pathology symptoms that correspond to the oncological disorder in humans. The subjects carry a regulatable transgene expression, of which is associated with tumor formation, and further carry regulatable genes for suppression of tumor formation. The disease-pathology symptoms are induced using a site-specific recombination system to induce expression of the transgene associated with tumor formation and negatively regulate or eliminate the genes for suppression of the tumor formation. The methods further involve analyzing tumor formation in subjects administered the compound and comparing appearance and amount of tumors in the subjects administered the compound with control subjects not administered the compound. Also included are a vector for engineering a disease model and a kit for its use.

What is claimed is: 
     
       1. A method for screening at least one compound to determine ability to treat an oncological disorder regulated through a tumor-inducing pathway, the method comprising:
 administering the compound to transgenic mice as an animal disease model, wherein the transgenic mice display pathology symptoms that correspond to the oncological disorder Glioblastoma multiforme in a human, wherein the genome of the transgenic mice comprises a regulatable transgene of human origin of a wild type epidermal growth factor receptor (EGFR WT ) and a mutant form vIII (EGFR vIII ) associated with tumor formation (EGFR WT / EGFR vIII ) or two copies of transgene EGFR vIII (EGFR vIII /EGFR vIII ), wherein expression of the transgene is associated with tumor formation, and the genome of the transgenic mice further comprises mutations in genes encoding Phosphatase and Tensin homolog detected on chromosome Ten (PTEN), p16Ink4a, and p19Arf as regulatable genes, wherein the disease pathology symptoms are induced using a-site-specific recombination system to induce expression of the transgene associated with tumor formation and negatively regulate or eliminate the gene expression of pTEN, p16Ink4a and p19Arf genes; 
 analyzing tumors appearing in the transgenic mice administered the compound; and, 
 comparing appearance and amount of tumors in the transgenic mice administered the compound and in control transgenic mice not administered the compound, wherein a decrease in tumors in the transgenic mice administered the compound compared to control transgenic mice is an indication that the compound treats the oncological disorder. 
 
     
     
       2. The method according to  claim 1 , wherein the transgene which encodes EGFR vIII  is under control of a cytomegalovirus (CMV) promoter immediate early enhancer and a chicken β-actin promoter sequence (pCAGGS), wherein the promoter is conditionally repressed by presence of a foxed stop cassette wherein the EGFR vIII  encoding region is flanked at 3′ and 5′ ends by collagen1α1 genomic sequences, and wherein the transgene is expressed in cells contacted with Cre recombinase. 
     
     
       3. The method according to  claim 2  further comprising performing stereotactic intracranial injection of adenovirus transducing Cre recombinase (Ad-CMVCre), wherein cre-lox function overproduces EGFR vIII  and EGFR WT  and down regulates Phosphatase and Tensin homolog detected on chromosome Ten (PTEN). 
     
     
       4. The method according to  claim 2 , further comprising observing an amount of expression of a bioluminescent marker under the control of a strong ubiquitous promoter, wherein the promoter is conditionally repressed by presence of the floxed stop cassette, wherein the marker is expressed in cells contacted with Cre recombinase, wherein the cells produce tumors and express the marker. 
     
     
       5. The method according to  claim 1 , wherein comparing appearance and the amount of the tumors is performed in live mice. 
     
     
       6. The method according to  claim 1 , wherein comparing the amount of the tumors further comprises observing by immunohistochemical staining of tumor sections from sacrificed mice or ex vivo mouse cell cultures at least one of: extent of cellularity as a measure of proliferation; presence of pleomorphic nuclei; presence of a fibrillary background; extent of membrane expression of EGFR; presence of astrocytic markers glial fibrillary acidic protein (GFAP) or S100β or both; extent of proliferating cells by presence of mitoses; extent of areas of necrosis; presence of perineuronal satellitosis;
 and presence of tumor cells migrated distal to main tumor mass. 
 
     
     
       7. The method according to  claim 1 , further comprising after comparing, analyzing an amount of mammalian target of rapamycin complex (mTORC) protein in the tumors in the transgenic mice administered the compound and in the control transgenic mice, wherein analyzing comprises determining at least one of: expression of mTORC per total protein; activation of mTORC activity; extent and pattern of mTORC phosphorylation; and relative usage of mTORC1 and mTORC2. 
     
     
       8. A method for screening at least one compound to determine ability to treat an oncological disorder regulated through a tumor-inducing pathway, the method comprising:
 administering the compound to transgenic mice as an animal disease model, wherein the transgenic mice display pathology symptoms that correspond to the oncological disorder Glioblastoma multiforme in a human; wherein the genome of the transgenic mice comprises a regulatable wild type EGFR (EGFR WT ) transgene of human origin and a transgene encoding human transforming growth factor-α(TGFα), wherein expression of the transgene EGFR WT  and the transgene TGFα are associated with tumor formation, and the genome of the transgenic mice further comprises mutations in genes encoding Phosphatase and Tensin homolog detected on chromosome Ten (PTEN), p16Ink4a, and p19Arf as regulatable genes, wherein the disease pathology symptoms are induced using a site-specific recombination system to induce expression of the transgene associated with tumor formation and negatively regulate or eliminate the gene expression of pTEN, p16Ink4a and p19Arf genes; 
 analyzing tumors appearing in the transgenic mice administered the compound; and, 
 comparing appearance and amount of tumors in the subjects administered the compound to that in control transgenic mice not administered the compound, wherein a decrease in tumors in the transgenic mice administered the compound compared to the control transgenic mice is an indication that the compound treats the oncological disorder. 
 
     
     
       9. The method according to  claim 8  further comprising regulating the tumor formation transgene with a cytomegalovirus (CMV) promoter immediate early enhancer and a chicken β-actin promoter sequence (pCAGGS), wherein the promoter is conditionally repressed by the presence of a floxed stop cassette, and wherein the transgene is flanked at 3′ and 5′ ends by collagen1α1 genomic sequences wherein the transgene is expressed in cells contacted with Cre recombinase. 
     
     
       10. The method according to  claim 8  further comprising performing stereotactic intracranial injection of adenovirus transducing Cre recombinase (Ad-CMVCre), wherein cre-lox function overproduces EGFR WT  and down regulates Phosphatase and Tensin homolog detected on chromosome Ten (PTEN). 
     
     
       11. The method according to  claim 8 , wherein comparing the appearance and the amount of the tumors in transgenic mice further comprises bioimaging to monitor tumor growth non-invasively. 
     
     
       12. The method according to  claim 11 , wherein non-invasive bioimaging comprises observing an amount of expression of a bioluminescent marker under the control of a strong ubiquitous promoter, wherein the promoter is conditionally repressed by presence of a floxed stop cassette, wherein the marker is expressed in cells contacted with Cre recombinase, wherein the cells produce tumors and express the marker. 
     
     
       13. The method according to  claim 12 , wherein comparing appearance and amount of tumors is performed in live mice. 
     
     
       14. The method according to  claim 8 , wherein comparing the amount of the tumors further comprises observing by immunohistochemical staining of tumor sections from sacrificed mice or ex vivo mouse cell cultures at least one of: extent of cellularity as a measure of proliferation; presence of pleomorphic nuclei; presence of a fibrillary background; extent of membrane expression of EGFR; presence of astrocytic markers glial fibrillary acidic protein (GFAP) or S100 β or both; extent of proliferating cells by presence of mitoses; extent of areas of necrosis; presence of perineuronal satellitosis; and presence of tumor cells migrated distal to main tumor mass.